Prophylaxis against smallpox and treatment of vaccinia with 1-methylisatin beta-thiosemicarbazone



United States Patent M 3 253,991 PROPHYLAXIS AGAINST SMALLPOX AND TREAT-MENT-OF VACCINIA WITH l-METHYLISATIN p-THIOSEMICARBAZONE Denis JohnBauer and Peter William adler, London, England, assignors to BurroughsWellcome & Co. (U.S.A.) Inc., Tuckahoe, N.Y., a corporation of New YorkNo Drawing. Filed Nov. 22, 1963, Ser. No. 325,790 3 Claims. (Cl. 167-65)This application is a continuation-in-part of application Serial No.73,559, filed December 5, 1960, now abandoned.

This invention relates to l-substituted isatin fl-thiosemicarbazones ofFormula I, and a process for the treatment of pox virus infectionscomprising the administration in therapeutically acceptable form thehost of such a l-substituted isatin fi-thiosemicarbazone of Formula I:

I 1'12 1'11 (I) wherein R is selected from the class consisting of theacetyl group, the alkyl, haloalkyl, hydroxyalkyl, alkenyl and alkynylgroups having not more than carbon atoms, and the lower aliphaticacyloxyalkyl groups in which the alkyl group has/not more than 5 carbonatoms, R is selected from the class consisting of'the hydrogen andhalogen atoms and the methyl and ethyl groups, and R and R may form at-rimethylene chain, and R and R are selected from the class consistingof the hydrogen and fluorine atoms, at least two of R R and R beinghydrogen atoms.

It has been found that certain l-substituted isatinfi-thiosemicarbazones of Formula I are highly active against infectionsof the vaccinia virus and the human smallpox (alastrim) virus in mice.Effective doses are well below the toxic level. Given orally to mice, adose of about 5 mg./kg. or less gives protection against alastrim orvaccinia, while mice survive doses of at least 1000 mg./ kg.

Especially active compounds include those of Formula I in which R is amethyl, ethyl, allyl or 2-hydroxyethyl group and R R and R are hydrogenatoms.

Certain compounds of Formula I, including those in which R is a methyl,allyl or acetoxymethyl group and R R and R are hydrogen atoms, appear tohave an advantage over other compounds of Formula I, including those inwhich R is an acetyl, ethyl, hydroxymethyl or Z-hydroxyethyl group and RR and R are hydrogen atoms, in that the former compounds exert avirustatic effect while the latter compounds may retard the growth ofvirus without themselves stopping it.

The compounds of Formula I are made by converting a l-substituted isatinof Formula II by known methods into its thiosemicarbazone.

3,253,991 Patented May 31, 1966 recrystallized from a solvent, forexample n-butanol.

For the treatment of pox virus infections, a compound of Formula I intherapeutically acceptable form is administered to the host. Thecompound may be presented in a pharmaceutical preparation,advantageously in discrete units, such as tablets, capsules, cachets,ampoules or suppositories, each containing a predetermined amount of thecompound. It may also be presented as a powder or granules, as asolution or suspension in an aqueous, non-aqueous or emulsified liquid,or as an ointment. For parenteral use, the preparations must be sterileand are presented in sea-led containers. The preparations of 'thisinvention may be made by any of the methods of pharrnacy, and mayinclude one or more of the following accessory ingredients: diluents,solutes, buffers, flavouring, binding, dispersing, surface-active,thickening, lubrieating, and coating materials, preservatives,antioxidants, bacteriostats, suppository and ointment bases, and anyother acceptable excipients.

It has also been discovered that the compounds of Formula I are moreactive against smallpox in man when 4 they are presented in a veryfinely divided form. The most useful compounds in this respect are thefollowing:

l-methylisatin fi-thiosemicarbazone, l-ethylisatin B-thiosemicarbazone,

l-allylisatin fi-thiosemicarbazone, l-hydroxymethylisatin,B-thiosemicarbazone, 1-2'-hydroxyethylisatin B-thiosemicarbazone,l-acetylisatin 3-thiosemicarbazone, S-fiuoro-l-methylisatinfi-thiosemicarbazone, l-ethyl-S-fluoroisatin ,B-thiosemicarbazone,1,7-tn'methyleneisatin fl-thiosemicarbazone, and 1,7-diethylisatinfi-thiosemicarbazone.

The finely divided compound of Formula I desirably has a surface areaper gram of at least 1.0 m. /g. (one square meter per gram), preferablyabout 24 'm. /g. The latter value corresponds to a particle size of 12,uwhen expressed as a surface mean diameter. (A definition of surface meandiameter is given in British Standard 2955; in general terms it is thediameter of the sphere of the material that would have the same surfacearea per gram.)

The surface area per gram of the finely divided compound of Formula Ican be measured by the usual techniques relying on well-known physicalphenomena. These include measurements determining particle size usingoptical or electron microscopy air permeability, sedimentation,elutriation, sieving, and electrical methods. Thus the surface area perparticle can be calculated and thence (from a knowledge of the densityof the material) the surface area per gram of the material.

When measuring the properties of crystalline and (especially) fibrousmaterials such as l-methylisatin flthiosemicarbazone, care must be takenthat the measurements obtained can be used to give a true indication ofthe surface area per gram of the material. For example, a sieve mayallow a long thin particle to pass through while retaining a more nearlyspherical particle of smaller individual volume and weight, andmeasurements of air permeability may be confused by a tortuosity factor.Microscopy, with statistical evaluation of the measured dimensions ofthe particles, is one of the most suitable methods for comparingdifferent physical forms.

The surface area per gram of a compound of Formula I may be increased byany of the usual techniques for obtaining material in a very finelydivided state.

For example, if the compound of Formula I is dissolved in a solvent inwhich it is readily soluble and this solution is rapidly poured intoanother liquid in which the compound is sparingly soluble, microcrystalsmay be formed.

Alternatively the coarser material obtained by the usual chemicalsynthesis can be comrninuted to a finer state of division, for exampleby means of a conventional ball, rod or tumbler mill (collectivelyreferred to as a cylinder mill). To be used effectively a cylinder millmust be studied and if necessary modified to take int-o account thephysical properties of the material actually being comminuted. Thus theamount of the material added to a mill of given dimensions, the numberand size of the rods or balls added, the speed of rotation of the mill,lubrication problems such as wet or dry milling, and considerations ofpressure and temperature (including cooling operations), may influencethe final state of division of the particles. The optimum grindingconditions for a given batch of a compound of Formula I are determinedby routine examination. Various modified forms of cylinder mill such asthe bi conical type, the spinning ball mill which exerts Coriolis forcesin addition to centrifugal and gravitational force, and many others mayalso be used. Another method of comminuting the material is in the fluidenergy mill which utilizes high velocity gaseous currents to enable theparticles to grind each other in the way that natural wind and streamsabrade rocks and sand down tofine dust. Yet another method of grindingutilizes vibration milling techniques; high frequency vibrations canproduce particles of extremely small dimensions.

Conventional separating techniques play a useful part in obtaining thedesired finely divided products. 'Thus sedimentation, sieving andelutriation techniques can be used to remove coarser particles, subject(as mentioned above) to due care with crystalline and fibrous materials.

Once the compound of Formula I has been prepared in a suitable formhaving the desired surface area per gram, it may be presented in any ofthe pharmaceutical preparations usually employed for presentinginsoluble drugs. Suitable preparations include granules, tablets,biscuits, syrups, capsules, fondants, pellets, suspensions, pastes anddraughts; and these may include the usual pharmaceutical excipients suchas diluents, wetting agents, flavoring agents, binders and effervescingagents. For example, the drug may be used in the form of a W./v. orw./v. suspension in syrup and dispensed in single-dose containers.

The dosage schedule for administering a compound of Formula I depends onthe body weight of the recipient (host) and also on the particular poxvirus infection to be treated. For the prophylactic treatment of humansmall pox contacts, for example, in order to prevent smallpox developingin persons Who have been in contact with the disease and. who wouldnormally be expected to contract it, the drug is preferably given in atleast two doses each in the range of 0.5 to 5.0 g.

In a particular example [D. J. Bauer, Leone St. Vincent, C. Henry Kempeand A. W. Downie, Prophylactic treatment of smallpox contacts withN-methylisatin B- thiosemicarbazone, The Lancet (September 7, 1963), 2,494-496]. l-methylisatin B-thiosemicarbazone was employed in dosageschedules of (a) 1.5 g. by mouth twice daily after meals for 4 days, (b)3.0 g. by mouth twice daily after meals for 4 days, and (c) two doses of3.0 g. by mouth taken after meals and within a period of 12 hours. Thisproved to be a very effective treatment of smallpox infections duringthe incubation period and thus protected smallpox contacts who had neverbeen vaccinated. In contacts who had had primary vaccination in the pastit was more effective than revaccination, and it was effective when thecontact was not detected soon enough for successful revaccination tooffer any chance of protection. The l-methylisatin fi-thiosemicarbazoneused in this study was a finely divided preparation having a surfacearea per gram of about 2-4 mP/g.

The following examples illustrate the invention.

Example 1.1-methylisatin fl-thiosemicarbazone 16.1 g. l-methylisatin and9.1 g. thiosemicarbazide were heated under reflux in 200 ml. 50% aqueousethanol for 30 minutes. The ethanol was removed by distillation invacuo, and the residue was cooled, filtered, washed with hot water, andrecrystallized from n-butanol to give the product, M.P. 245 C.

Example 2.Ethylisatin fl-thiosemicarbazone 23 g. sodium was dissolved inone liter of absolute ethanol, 147 g. isatin was added with shaking, andthe mixture was heated under reflux for 15 minutes. 308 g. ethyl iodidewas added, and heating under reflux continued for a further 12 hours.Ethanol and ethyl iodide were removed in vacuo. The residue wasdissolved by heating under reflux in 2 liters of benzene, the solutionwas extracted with 2 liters of N-sodium hydroxide in two portions, andthe alkaline solution was adjusted to pH 1 by addition of concentratedhydrochloric acid, when an oil separated. This was extracted with 2liters of benzene in portions. The benzene solution was dried overanhydrous sodium sulfate, and the benzene removed in vacuo.

The residue (l-ethylisatin) was dissolved by heating under reflux in 500ml. ethanol. A solution of 91 g. thiosemicarbazide in 500 ml. hot waterwas added, and the mixture heatedunder reflux for 30 minutes. Ethanolwas removed in vacuo, and the residue was cooled and shaken to solidfythe product. This was filtered off and washed with hot water, suspendedin 2 liters of distilled water at C. with rapid stirring for 15 minutes,filtered oif, and washed with distilled waterh The residue wascrystallized from 3 litres of n-propanoi, decolorizing with charcoal, togive 150 g. l-ethylisatin B-thiosemicarbazone, M.P. 204 C.

Example 3.1n-pr0pylisatin flrthiosemiearbazone The fi-thiosemicarbazone,M.P. 193 C., was prepared from l-n-propylisatin in a manner similar tothat described in Example 1.

Example 4.1-n-butylisatin ,B-thiosemiearbazone Equivalent molecularproportions of l-sodioisati'n and n-butyl bromide were refluxed inbenzene for 10 days. The liquid was filtered and the filtrate extractedwith 2 N-sodium hydroxide. The extract was acidified with concentratedhydrochloric acid to give l-n-busylisatin as an orange oil whichsolidified on standing, M.P. 36 C. The B-thiosemicarbazone, M.P. C., wasprepared from the isatin in a manner similar to that described inExample 1.

Example 5.-1-n-pentylisatin ,B-thiosemicarbazone Equivalent molecularproportions of l-sodioisatin and n-pentyl bromide were heated underreflux for 48 hours. The mixture was cooled and filtered. The filtratewas extracted with a small volume of 2 N-sodium hydroxide, andacidification of the alkaline extract with concentrated hydrochloricacid gave l-n-pentylisatin, M.P. 47 C. The B-thiosemicarbazone, M.P. 184C., was prepared from the isatin in a manner similar to that describedin Example 1.

Example 6.-1-is0pr0pylisatin fi-thiosemicarbazone l-isopropylisatin,M.P. about 62 C. (diflicult to crystallize), and its,B-thioseniicarbazone, M.P. 225 C., were similarly prepared.

Example 7.]-pr0p-2'-ynylisatin fi-thiosemicarbazone 1-prop-2-ynylisatin,M.P. 163 C., and its fi-thiosemicarbazone, M.P. 250 C. (decomp.), weresimilarly prepared.

Example 8.1-allylisatin fi-thiosemz'carbazone l-sodioisatin from 7.5 g.isatin and allyl bromide were heated under reflux in xylene for 2 hours.The mixture was filtered and extracted with 2 N-sodium hydroxide. Theextract was cleared by ether extraction and treated with hydrochloricacid to precipitate red crystals of l-allylisatin, M.P. 89 C.

This isatin was treated with an equivalent molecular proportion ofthiosemicarbazide using the procedure of Example 1. The productcrystallized from anhydrous ethanol in orange crystals, M.P. 201 C.

Example 9.-1-hydr0xymethylisatin fi-thiosemicarbazone TheB-thiosemicarbazone, M.P. 230 C., was prepared froml-hydroxymethylisatin in a manner similar to that described in Example1.

Example 10.] -/3-hydroxyethylisatin 2-thiosemicarbaz0ne Equivalentmolecular proportions of l-sodioisatin and 2-chloroethanol were heatedunder reflux for 48 hours. The mixture was cooled and filtered. Thefiltrate was reduced to small volume, and 1-2-hydroxyethylisatinseparated on addition of petroleum ether (B.P. 6080 It crystallized fromaqueous methanol in orange needles, M.P. 118 C.

The isatin was treated with an equivalent molecular proportion ofthiosemicarbazide using the procedure of Example 1. The productcrystallized from aqueous methanol in bright orange needles, M.P. 247 C.

Example 11.-1-acet0xymethylisatin ,B-thiosemicarbazone tallized fromn-butanol.

Example 12.-1-2'-acet0xyethylisatin B-th iosem i carbazone1-2'-acetoxyethylisatin, M.P. 113 C., and its thiosemicarbazone, M.P.244 C. (decomp) when crystallized' from aqueous ethanol, were similarlyprepared from 1-2- hydroxyethylisatin.

Example 13.-1-2'-bromoethylisatin B-thiosemicarbazone Example14.1-aeetylisatin ,B-tlziosemicarbazone The ,B-thiosemicarbazone, M.P.244 C., was prepared from l-aeetylisatin in a manner similar to thatdescribed in Example 1.

Example I5.5-flu0r0-]-methylisatin ,B-thiosemicarbazone Equimolarquantities of S-fluoro-l-sodioisatin and methyl iodide were heated underreflux in ethanol for 48 hours. The mixture was filtered and thefiltrate extracted with benzene. The benzene solution was extracted with2 N-sodium hydroxide. Acidification with concentrated hydrochloric acidof the alkaline extract gave S-fluoro-bmethylisatin as light redneedles, M.P. 151 C. The fl-thiosemicarbazone, M.P. 260 C. whencrystallized from ethanol, was prepared from the isatin in a mannersimilar to that described in Example 1.

.methylisatin,

6 Example 16. 1-ethyl-5-flu0r0isatin fl-lhiosemicarbazone1-ethyl-5-fluoroisatin, red needles, M.P. 131 C. and its,B-thiosemicarbazone, orange needles, M.P. 228 C., were preparedsimilarly from S-fluoro-l-sodioisatin.

Example 17.1,7-trimelhyleneisatin fl-thiosemicarbazone Equimolarquantities of 1,7-trimethyleneisation (red plates, M.P. 191 C.) andthiosemicarbazide were heated under reflux in aqueous ethanol. Theproduct was 001- lected and washed well with hot water, and crystallizedfrom ethanol as orange plates, M.P. 236 C.

Example 18.-1,7-a'iethylisatin fi-thiosemicarbazone Example 19.Tabletsl-ethylisatin fi-thiosemicarfbazone (250 parts) was triturated until thecrystals were finely powdered, mixed with lactose (50 parts) and starch(30 parts), and granulated with 5% starchmucilage or with 10% aqueousalcohol gelatin solution. The moist mass was sifted through a sievehaving 7.9 meshes/cm. The granules were dried at about 50 C. Magnesiumstearate (1.5 parts) was added. The mixture was sifted through a sievehaving 6.3 meshes/ cm. and compressed on a suitable die to give tabletseach containing 250 mg. of 1- ethylisatin fi-thiosemicarbazone. Tabletscontaining the p-thiosemicarbazone of l-methylisatin, l-allylisatin,1-2' hydroxyethylisatin, l-acetoxymethylisatin or1-2'-acetoxyethylisatin were similarly prepared.

Example 20.Capsules Hard gelatin capsules were each filled with 250 mg.of the finely powdered B-thiosemicarbazone of l-methylisatin,l-ethylisatin, l-n-propylisatin, l-isopropylisatin, l-n-butylisatin,l-n-pentylisatin, l-allylisatin, 1-prop-2- ynylisatin,l-hydroxymethylisatin, 1-2'-hydroxyethylisatin, l-acetoxymethylisatin,1-2-acetoxyethylisatin, 1-2'-bromoethylisatin, l-acetylisatin,S-iuoro-l-methylisatin, 1- ethyl-S-fiuoroisatin, 1,7-trimethyleneisatinor 1,7-dietl1ylisatin.

Example 21.Capsules The dried granules, prepared in Example 19 andcontaining either l-ethylisatin fi-thiosemicarbazone or 1- methylisatinfi-thiosemicarbazone were filled by means of a iburette into hardgelatin capsules; each to contain 250 mg. of the active ingredient.

Example 22.Aque0us suspension for intramuscular injection l-ethylisatin,B-thiosemicarbazone was sterilized by heatng at 160 C. for one hour. Asterile aqueous vehicle was made by autoclaving the following solution:benzyl alcohol (2%), sodium carlboxymethylcellulose (2%),polyoxyethylene sorbitan monooleate (0.1%) and water for injection (toUsing aseptic precautions throughout, the sterile drug (20%) was mixedwith the sterile aqueous vehicle, and a fine suspension was preparedusing a sterile ball mill.

Aqueous suspensions of the B-thiosemicarbazone of 1- methylisatin,l-n-propylisatin, l-isopropylisatin, l-n-pentylisatin, l-allylisatin,1-prop-2'-ynylisatin, l-hydroxy- 1-2'-hydroxyethylisatin,l-acetoxymethylisatin, 1-2'-acetoxyethylisatin, l-acetylisatin,S-fluoro-lmethylisatin, l-ethyl-S-fluoroisatin, 1.7-trimethyleneisatinor 1,7-diethylisatin were similarly prepared.

sterilized by heating for an hour at C. in thin layers in a coveredvessel, and arachis oil was sterilized by heating to 106 C. for onehour. Using aseptic precautions throughout, a suspension containing 200mg. drug/ml.

was prepared as follows: the sterile arachis oil was added to thesterile drug in a sterile porcelain ball mill provided with porcelainballs, the mixture was milled until the drug was suitaby suspended inthe 'arachis oil, and the suspension was transferred to suitable sterileampoules or multidose containers which were sealed in the usual manner.

A similar suspension of l-rnethylisatin B-thiosemicarbazone was preparedin the same way.

Example 24.Sterile eye ointment Finely powdered l-ethylisatinfi-thiosemicarbazone was sterilized by heating to 160 C. in thin layersfor one hour. An ointment containing 5% of drug was prepared by mixingthe sterile drug with sterile molten eye ointment basis (BritishPharmacopoeia) and stirring aseptically until cool. The medicated eyeointment was packed in 4 g. lots in small sterilized collapsible tulhes.

A similar ointment was made by triturating the sterile drug in a sterilemortar with a little sterile liquid paraflin, and then adding thesterile molten eye ointment basis and proceeding as described above.

Similar ointments were made containing the B-thiosemicarbazone ofl-methylisatin, l-allylisatin, 1-2-hydroxyethylisatin,l-acetoxymethylisatin or 1-2-acetoxyethy1- isatin.

Example 25.-Micrcrystals A 5% w./v. solution of l-methylisatinfi-thiosemicarbazone in dimethylformamide (20 ml.) was poured into vwater (80 ml.) to give a thick and stable suspension of microcrystalshaving a length of about 20-25a and diameter up to La.

Example 26.C0mminuted material l-methylisatin B-thiosemicarbazone wasdry-milled for a period of days in a conventional rod mill until theparticles had a surface mean diameter of 12,a (corresponding to asurface area per gram of 2-4 'm. /g.).

Example 27.Suspension in syrup A 10% w./v. suspension of l-methylisatinfi-thiosemicarbazone in syrup was prepared by the usual pharmaceuticaltechniques according to the following formula:

Purified water, to 100.0 ml.

Example 28.Capsules A chewable gelatin capsule was prepared by mixing asyrup according to the following formula:

Finely divided l-methylisatin B-thiosemicarbazone (2-4 mP/g.) 33.33Methyl p-hydroxybenzoate 0.1 Flavoring agents such as vanilla, quantumsuflicit 70% w./v. sorbitol syrup, to 100.0

Portions of this syrup were then encapsulated using a flavored,chewable, soft gelatin base, so that each capsule contained 1.5. g. ofl-methylisatin fi-thiosemicarbazone.

Example 29.-F0na'ants Fondants in the form of cubes approximately 13 mm.across were prepared by the usual pharmaceutical techniques according tothe following formula:

G. Finely divided l-methylisatin p-thiosemicarbazone (2-4 m. /g.) 40.0Glycerol 7.0 Flavoring agents such as apricot or pineapple syntheticflavoring, quantum sufficit Fondant base, to 100.0

References Cited by the Examiner UNITED STATES PATENTS 2,642,439 6/1953Coles 260325 3,076,815 2/1963 Pugin 260325 3,086,911 4/1963 Brown et a1.167-65 3,088,871 5/1963 PfeiiTer 167-65 OTHER REFERENCES Bauer, D. J.,et al.: Antiviral Chemotherapeutic Activity ofIsatin-3-Thiosemicarbazone in Mice Infected With Rabbit Pox Virus,Nature, 184, supple. 19, pages 1496- 7, Nov. 7, 1959.

Bauer, D. 1.: Antiviral and Synergistic Actions of IsatinThiosem-icarbazone and Certain Phenoxypyrimidines in Vaccinia Infectionin Mice, Brit. J. Exptl. Pathol, 36, pp. -114 (1955).

Bock, M.: The Action of Thiosemicarbazone on the Experimental SmallpoxInfection of the Mouse, Z. Hyg. Infektionskrankh, 143, pp. 480-9 (1957);abstracted in English in Chem. Abstracts, 51, 10748C (1957).

Minton, S. A.,-et al.: EffectotlThiosemicarbazones andDichlorophenoxythiouracil on Multiplication of a Recently IsolatedStrain of Variola-Vaccinia Virus in the Brain of the Mouse, J. Immunol.,70, pp. 222-8 (1953); abstracted in Chem. Abstracts, 47, 5542B (1953).

Thompson, R. L., et al.: Effect of Aliphatic Oximes and IsatinThiosemicarbazones on Vaccinia Infection in the Mouse and Rabbit, Proc.Soc. Exptl. Biol, Med., 84, pp. 496-9, 1953.

LEWIS GOTTS, Primary Examiner.

NICHOLAS S. RIZZO, Examiner.

MARY U. QBRIEN, SHEP K. ROSE,

Assistant Examiners.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION atent No.3,253,991 May 31, 1966 Denis John Bauer et a1.

It is hereby certified that error appears in the above numbered patantrequiring correction and that the said Letters Patent should read as:orrected below.

Column 1, line 17, for "form" read form to column I, line 8, for"Ethylisatin", in italics, read l-Ethy- .isatin in italics; line 48, for"l-n-busylisatin" read l-n-butylisatin column 5, line 17, for"lg'-hydroxy :thylisatin", in italics read l 2 hydroxyethylisatin .nitalics; line 18, for "Z-thiosemicarbazone", in italics, 'ead6-thiosemicarbazone in italics column 6 line for"l,7-trimethyleneisation" read 1,7-trimethyleneiatin line 54, for"heatng" read heating line 67 'or "l.7-trimethyleneisatin" read1,7-trimethyleneisatin Signed and sealed this 29th day of October 1968.

SEAL] ttest:

DWARD M.FLETCHER,JR. EDWARD J. BRENNER ttesting Officer Commissioner ofPatents

1. A PROCESS FOR THE PROPHYLAXIS AGAINST SMALLPOX AND TREATMENT OFINFECTIVE COMPLICATIONS AND MODIFICATION OF SEVERE VACCINATION REACTIONVACCINA WHICH COMPRISES THE ADMINISTRATION IN THERAPEUTICALLY ACCPTABLEFORM TO THE HOST OF A THERAPEUTICALLY EFFECTIVE DOSAGE OF 1-METHYLISATINB-THIOSEMICARBAZONE.